Novel inhibitors of rho kinase

ABSTRACT

The present invention relates to compounds and methods which may be useful as inhibitors of Rho kinase for the treatment or prevention of disease.

This application claims the benefit of priority of U.S. provisionalapplication No. 60/832,346, filed Jul. 20, 2006, the disclosure of whichis hereby incorporated by reference as if written herein in itsentirety.

The present invention is directed to new pyridine and benzothiophenecompounds and compositions and their application as pharmaceuticals forthe treatment of disease. Methods of inhibition of Rho kinase activityin a human or animal subject are also provided for the treatment ofdiseases such as ophthalmologic diseases.

Many cell signaling events activate one or more members of the smallmonomeric GTPase superfamily. The Rho subfamily of GTPases (consistingof RhoA, RhoB, and RhoC) transmits signals, frequently from cell surfacereceptors, to effectors that play critical roles in control ofcytoskeletal dynamics and gene regulation [Ridley, A. J., 2001, TrendsCell Biol. 11:471-477; Jaffe, A. B. and Hall, A., 2005, Annu Rev CellDev Biol. 21:247-269]. In particular, Rho-mediated effects on thecytoskeleton influence non-muscle cell shape, smooth muscle cellcontraction, cell-cell and cell-matrix adhesion, intracellular vesicletransport, axonal and dendrite growth, vascular architecture, immune andinflammatory cell migration, and cleavage furrow formation and functionduring cell division [Bussey, H., 1996, Science. 272:224-225; Fukata, Y.et al., 2001, Trends Pharmacol Sci. 22:32-39; Luo, L., 2000, Nat RevNeurosci. 1:173-180; Hu, E. and Lee, D., 2003, Curr Opin Investig Drugs.4:1065-1075; Bokoch, G. M. 2005, Trends Cell Biol. 15:163-171;Wadsworth, P., 2005, Curr Biol. 15:R871-874].

Although the Rho GTPase cycle is complex, it can be briefly summarizedas follows. Inactive, GDP-bound Rho, complexed with a GDP dissociationinhibitor protein (GDI), is recruited to the plasma membrane in responseto signaling events, such as ligand binding to cell surface receptors.The GDI is displaced, whereby the inactive GDP-bound Rho is converted toactive GTP-bound Rho by membrane-localized guanine-nucleotide exchangefactors. GTP-bound Rho then binds and activates a number of effectors atthe plasma membrane. Many proteins controlled by Rho activity have beenidentified, including a variety of protein and lipid kinases [Kaibuchi,K. et al., 1999, Annu Rev Biochem. 68:459-486; Bishop, A. L. and Hall,A., 2000, Biochem J. 348:241-255]. The intrinsic GTPase activity of Rho,stimulated by GTPase activating proteins, converts Rho back to theinactive, GDP-bound form, whereupon GDP-bound Rho can be extracted fromthe plasma membrane by the GDI (although in some instances, the GDI mayextract GTP-bound Rho to extinguish a signal, or redirect GTP-bound Rhoto a different compartment) [Sasaki T., and Takai Y., 1998, BiochemBiophys Res Commun. 245:641-645; Olofsson, B., 1999, Cell Signal.11:545-554; Schmidt, A. and Hall, A., 2002, Genes Dev. 16:1587-1609;Moon, S. Y. and Zheng, Y., 2003, Trends Cell Biol. 13:13-22].

Of identified Rho effectors, the Rho-associated coiled-coil containingkinases, here referred to as Rho kinases, have been the subject ofintense investigation in molecular and cell biological studies, and aspharmaceutical targets in multiple therapeutic areas. Rho kinases areserine-threonine protein kinases of approximately 160 kD molecularweight that contain an amino-terminal kinase catalytic domain, a longamphipathic alpha helical (coiled-coil) domain, an activated Rho bindingdomain, and a carboxy-terminal pleckstrin-homology domain (promotingbinding to plasma membrane phosphoinositides) that is split by acysteine rich zinc-finger like motif [Ishizaki, T., et al., 1996, EMBOJ. 15, 1885-1893; Fujisawa, K. et al., 1996, J Biol Chem.271:23022-23028; Matsui, T. et al., 1996, EMBO J. 15:2208-2216]. Thereare two known isoforms of Rho kinase, although splice variants mayexist. These isoforms are referred to as Rho kinase (ROK) alpha(referred to here as ROCK2), and Rho kinase (ROK) beta, also known asp160 ROCK (referred to here as ROCK1) [Leung, T. et al., 1996, Mol CellBiol. 16:5313-5327; Nakagawa, O. et al., 1996, FEBS Lett. 392:189-193].Many protein kinases are controlled by reversible phosphorylation eventsthat switch them between active and inactive states. By contrast, Rhokinases switch from low, basal activity to high activity by reversiblebinding to GTP-bound Rho. Active Rho kinases then phosphorylateadditional effectors of Rho signaling in the vicinity of the plasmamembrane. Both Rho kinases are expressed in a mostly ubiquitous fashionin mammalian tissues at low to moderate levels, although expression ishighly enriched in some cell types. Rho kinases share functionalhomology in their catalytic domains with the protein kinase A and Cfamilies, and a variety of small molecule inhibitors of Rho kinases alsobind and inhibit protein kinase A in particular [Breitenlechner, C. etal., 2003, Structure. 11:1595-1607]. ROCK1 has 64% sequence identity toROCK2 throughout the protein structure, and the kinase domains arehighly conserved (90% identical).

As effectors of Rho signaling, Rho kinases are directly involved incontrolling cytoskeleton dynamics, gene regulation, cell proliferation,cell division, and cell survival. Constitutively active mutants of Rhokinases can be generated by truncating carboxy-terminal regions, as faras the kinase domain, suggesting important negative regulation by thecarboxy-terminal sequences. Expressed in cells, these mutants generatephenotypes consistent with hyperactive Rho kinase activity (e.g.increased stress fiber formation and cell-substrate focal adhesions). Bycontrast, deletion of the catalytic domain of Rho kinases results in atrans-dominant inhibitory effect in cells [Amano, M. et al., 1997,Science. 275:1308-1311; Leung, T. et al., 1996, Mol Cell Biol.16:5313-5327; Amano, M. et al., 1999, J Biol Chem. 274:32418-32424].There is data consistent with separable functions for ROCK1 and ROCK2 incells, although these observations may be cell-type specific [Yoneda, A.et al., 2005, J Cell Biol. 170:443-453]. Although genetic knockout ofROCK1 leads to perinatal lethality due to omphaloceles in newborns, andgenetic knockout of ROCK2 leads to a high incidence of embryoniclethality due to poor placental development, neither knockout alone isconsistent with the necessity of ROCK1 or ROCK2 for most normal cellbehaviors of the embryo during development [Shimizu, Y. et al., 2005, JCell Biol. 168:941-953; Thumkeo, D. et al., 2003, Mol Cell Biol.23:5043-5055].

Rho kinases can phosphorylate a variety of substrates to control variousaspects of cytoskeletal behavior [Riento, K. and Ridley, A. J. 2003, NatRev Mol Cell Biol. 4:446-456]. Many studies have focused on control ofthe myosin light chain (MLC) regulatory subunit. Phosphorylation of theMLC regulatory subunit leads to increased actomyosin activity (e.g.smooth muscle cell contraction or increased non-muscle cell stressfibers). Rho kinases stimulate actomyosin activity by directphosphorylation of the MLC regulatory subunit, and by inactivation ofmyosin light chain phosphatase through the phosphorylation of its myosinbinding subunit [Amano, M. et al., 1996, J Biol Chem. 271:20246-20249;Kimura, K. et al., 1996, Science. 273:245-248; Kureishi, Y. et al.,1997, J Biol Chem. 272:12257-12260]. LIM kinase, ezrin/radixin/moesin(ERM) family proteins, and adducin are some additional substrates of Rhokinases, and the phosphorylation of these and other proteins altersvarious aspects of cytoskeletal function [Oshiro, N., et al., 1998, JBiol Chem. 273:34663-34666; Kimura, K., et al., 1998, J Biol Chem.273:5542-5548; Matsui, T., et al., 1998, J Cell Biol. 140:647-657;Fukata, Y., et al., 1999, J Cell Biol. 145:347-361; Kosako, H., et al.,1997, J Biol Chem. 272:10333-10336; Goto, H., et al., 1998, J Biol Chem.273:11728-11736; Maekawa, M., et al., 1999, Science. 285:895-898;Ohashi, K., et al., 2000, J Biol Chem. 275:3577-3582].

Small molecule compounds such as Y-27632, Y-32885, Y-39983, HA-1077(fasudil), hydroxy-fasudil, and a dimethylated analog of fasudil(H-1152P, or HMN-1152) have been demonstrated to directly inhibit Rhokinases. The Y compounds, which are more selective Rho kinaseinhibitors, contain a common pyridine moiety, while fasudil and itsanalogs contain a common isoquinoline scaffold. Crystal structures forthe kinase domain of ROCK1 complexed with Y-27632, fasudil,hydroxy-fasudil, and H-1152P have been reported (Jacobs, M. et al.,2006, J Biol Chem. 281:260-268]. All of these compounds occupy part ofthe ATP-binding pocket, consistent with the fact that they arereversible ATP competitive inhibitors.

These same Rho kinase inhibitors are cell permeable, and cause changesin cytoskeletal function and cell behavior consistent with loss of Rhokinase activity, similar to effects of the trans-dominant inhibitorymutants. Effects have been observed both in cultured cells in vitro andin physiologically responsive tissues in vivo [Nagumo, H. et al., 2000,Am J Physiol Cell Physiol. 278:C57-C65; Sinnett-Smith, J. et al., 2001,Exp Cell Res. 266:292-302; Chrissobolis, S. and Sobey, C. G., 2001, CircRes. 88:774-779; Honjo, M. et al., 2001, Invest Ophthalmol Vis Sci.42:137-144; Takahara, A. et al., 2003, Eur J Pharmacol. 460:51-57;Fournier, A. E. et al., 2003, J Neurosci. 23:1416-1423; Rikitake, Y. etal., 2005, Stroke. 36:2251-2257; Slotta, J. E. et al. 2006, Inflamm Res.55:364-367; Ying, H. et al., 2006, Mol Cancer Ther. 5:2158-2164]. Thecorrelation between small molecule inhibition of Rho kinases and changesin cell behavior both in vitro and in vivo (e.g., vascular smooth musclerelaxation, bronchial smooth muscle relaxation, inhibition of immune andinflammatory cell migration, inhibition of tumor cell migration,inhibition of experimentally induced fibrosis, promotion of neuralregenerative activity) supports the notion that Rho kinases aresignificant pharmaceutical targets for a wide range of therapeuticindications. In addition, it is now more appreciated that some of the“pleiotropic” and beneficial cardiovascular effects of clinically usefulHMG Coenzyme A reductase inhibitors (i.e., the “statin” drug class) area consequence of decreased Rho, and therefore decreased Rho kinase,activity, especially in endothelial cells [Eto, M. et al., 2002,Circulation. 105:1756-1759; Rikitake, Y. and Liao, J. K., 2005, CircRes. 97:1232-1235; Kozai, T. et al., 2005, Cardiovasc Res. 68:475-482;Girgis, R. E. et al., 2007, Am J Physiol Lung Cell Mol Physiol.292:L1105-L1110]. Interestingly, Rho kinase inhibition has been recentlyimplicated in the enhanced survival and cloning efficiency ofdissociated human embryonic stem cells, which suggests the utility ofRho kinase inhibitors for stem cell therapies [Watanabe, K. et al.,2007, Nat Biotechnol. 25:681-686].

Compounds and pharmaceutical compositions, certain of which have beenfound to inhibit Rho kinase, have been discovered, together with methodsof using the compounds including methods for the treatment of Rhokinase-mediated diseases in a patient by administering the compounds.

Certain compounds according to the present invention possess useful Rhokinase inhibiting activity, and may be used in the treatment orprophylaxis of a disease or condition in which Rho kinase plays anactive role. Thus, in broad aspect, the certain embodiments of thepresent invention also provide pharmaceutical compositions comprisingone or more compounds disclosed herein together with a pharmaceuticallyacceptable carrier, as well as methods of making and using the compoundsand compositions. Certain embodiments of the present invention providemethods for inhibiting Rho kinase. Other embodiments of the presentinvention provide methods for treating a Rho kinase-mediated disorder ina patient in need of such treatment, comprising administering to saidpatient a therapeutically effective amount of a compound or compositionaccording to the present invention. The present invention alsocontemplates the use of certain compounds disclosed herein for use inthe manufacture of a medicament for the treatment of a disease orcondition ameliorated by the inhibition Rho kinase.

In certain further embodiments, the compounds of the present inventionmay find use in the inhibition of Rho kinase for the treatment ofdisease.

In certain yet further embodiments, the compounds of the presentinvention may be administered in combination with at least one othertherapeutic agent.

As used herein, the terms below have the meanings indicated.

When ranges of values are disclosed, and the notation “from n₁ . . . ton₂” is used, where n₁ and n₂ are the numbers, then unless otherwisespecified, this notation is intended to include the numbers themselvesand the range between them. This range may be integral or continuousbetween and including the end values. By way of example, the range “from2 to 6 carbons” is intended to include two, three, four, five, and sixcarbons, since carbons come in integer units. Compare, by way ofexample, the range “from 1 to 3 μM (micromolar),” which is intended toinclude 1 μM, 3 μM, and everything in between to any number ofsignificant figures (e.g., 1.255 μM, 2.1 μM, 2.9999 μM, etc.).

The term “about,” as used herein, is intended to qualify the numericalvalues which it modifies, denoting such a value as variable within amargin of error. When no particular margin of error, such as a standarddeviation to a mean value given in a chart or table of data, is recited,the term “about” should be understood to mean that range which wouldencompass the recited value and the range which would be included byrounding up or down to that figure as well, taking into accountsignificant figures.

The term “acyl,” as used herein, alone or in combination, refers to acarbonyl attached to an alkenyl, alkyl, aryl, cycloalkyl, heteroaryl,heterocycloalkyl, or any other moiety were the atom attached to thecarbonyl is carbon. An “acetyl” group, which is a type of acyl, refersto a —C(O)CH₃ group. An “alkylcarbonyl” or “alkanoyl” group refers to analkyl group attached to the parent molecular moiety through a carbonylgroup. Examples of such groups include methylcarbonyl and ethylcarbonyl.Examples of acyl groups include formyl, alkanoyl and aroyl.

The term “alkenyl,” as used herein, alone or in combination, refers to astraight-chain or branched-chain hydrocarbon radical having one or moredouble bonds and containing from 2 to 20 carbon atoms. In certainembodiments, said alkenyl will comprise from 2 to 6 carbon atoms. Theterm “alkenylene” refers to a carbon-carbon double bond system attachedat two or more positions such as ethenylene [(—CH═CH—),(—C::C—)].Examples of suitable alkenyl radicals include ethenyl, propenyl,2-methylpropenyl, 1,4-butadienyl and the like. Unless otherwisespecified, the term “alkenyl” may include “alkenylene” groups.

The term “alkoxy,” as used herein, alone or in combination, refers to analkyl ether radical, wherein the term alkyl is as defined below.Examples of suitable alkyl ether radicals include methoxy, ethoxy,n-propoxy, isopropoxy, n-butoxy, iso-butoxy, sec-butoxy, tert-butoxy,and the like.

The term “alkyl,” as used herein, alone or in combination, refers to astraight-chain or branched-chain alkyl radical containing from 1 to 20carbon atoms. In certain embodiments, said alkyl will comprise from 1 to10 carbon atoms. In further embodiments, said alkyl will comprise from 1to 6 carbon atoms. Alkyl groups may be optionally substituted as definedherein. Examples of alkyl radicals include methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl,hexyl, octyl, noyl and the like. The term “alkylene,” as used herein,alone or in combination, refers to a saturated aliphatic group derivedfrom a straight or branched chain saturated hydrocarbon attached at twoor more positions, such as methylene (—CH₂—). Unless otherwisespecified, the term “alkyl” may include “alkylene” groups.

The term “alkylamino,” as used herein, alone or in combination, refersto an alkyl group attached to the parent molecular moiety through anamino group. Suitable alkylamino groups may be mono- or dialkylated,forming groups such as, for example, N-methylamino, N-ethylamino,N,N-dimethylamino, N,N-ethylmethylamino and the like.

The term “alkylidene,” as used herein, alone or in combination, refersto an alkenyl group in which one carbon atom of the carbon-carbon doublebond belongs to the moiety to which the alkenyl group is attached.

The term “alkylthio,” as used herein, alone or in combination, refers toan alkyl thioether (R—S—) radical wherein the term alkyl is as definedabove and wherein the sulfur may be singly or doubly oxidized. Examplesof suitable alkyl thioether radicals include methylthio, ethylthio,n-propylthio, isopropylthio, n-butylthio, iso-butylthio, sec-butylthio,tert-butylthio, methanesulfonyl, ethanesulfinyl, and the like.

The term “alkynyl,” as used herein, alone or in combination, refers to astraight-chain or branched chain hydrocarbon radical having one or moretriple bonds and containing from 2 to 20 carbon atoms. In certainembodiments, said alkynyl comprises from 2 to 6 carbon atoms. In furtherembodiments, said alkynyl comprises from 2 to 4 carbon atoms. The term“alkynylene” refers to a carbon-carbon triple bond attached at twopositions such as ethynylene (—C:::C—, —C≡C—). Examples of alkynylradicals include ethynyl, propynyl, hydroxypropynyl, butyn-1-yl,butyn-2-yl, pentyn-1-yl, 3-methylbutyn-1-yl, hexyn-2-yl, and the like.Unless otherwise specified, the term “alkynyl” may include “alkynylene”groups.

The terms “amido” and “carbamoyl,” as used herein, alone or incombination, refer to an amino group as described below attached to theparent molecular moiety through a carbonyl group, or vice versa. Theterm “C-amido” as used herein, alone or in combination, refers to a—C(═O)—N(R)₂ group with R as defined herein. The term “N-amido” as usedherein, alone or in combination, refers to a RC(═O)N(R′)— group, with Rand R′ as defined herein. The term “acylamino” as used herein, alone orin combination, embraces an acyl group attached to the parent moietythrough an amino group. An example of an “acylamino” group isacetylamino (CH₃C(O)NH—).

The term “amino,” as used herein, alone or in combination, refers to—N(R)(R′) or —N⁺(R)(R′)(R″), wherein R, R′ and R″ are independentlyselected from the group consisting of hydrogen, alkyl, acyl,heteroalkyl, aryl, cycloalkyl, heteroaryl, and heterocycloalkyl, any ofwhich may themselves be optionally substituted.

The term “amino acid,” as used herein, alone or in combination, means asubstituent of the form —NRCH(R′)C(O)OH, wherein R is typicallyhydrogen, but may be cyclized with N (for example, as in the case of theamino acid proline), and R′ is selected from the group consisting ofhydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl,heteroaryl, amino, amido, cycloalkylalkyl, heterocycloalkylalkyl,arylalkyl, heteroarylalkyl, aminoalkyl, amidoalkyl, hydroxyalkyl, thiol,thioalkyl, alkylthioalkyl, and alkylthio, any of which may be optionallysubstituted. The term “amino acid” includes all naturally occurringamino acids as well as synthetic analogues.

The term “aryl,” as used herein, alone or in combination, means acarbocyclic aromatic system containing one, two or three rings whereinsuch rings may be attached together in a pendent manner or may be fused.The term “aryl” embraces aromatic radicals such as benzyl, phenyl,naphthyl, anthracenyl, phenanthryl, indanyl, indenyl, annulenyl,azulenyl, tetrahydronaphthyl, and biphenyl.

The term “arylalkenyl” or “aralkenyl,” as used herein, alone or incombination, refers to an aryl group attached to the parent molecularmoiety through an alkenyl group.

The term “arylalkoxy” or “aralkoxy,” as used herein, alone or incombination, refers to an aryl group attached to the parent molecularmoiety through an alkoxy group.

The term “arylalkyl” or “aralkyl,” as used herein, alone or incombination, refers to an aryl group attached to the parent molecularmoiety through an alkyl group.

The term “arylalkynyl” or “aralkynyl,” as used herein, alone or incombination, refers to an aryl group attached to the parent molecularmoiety through an alkynyl group.

The term “arylalkanoyl” or “aralkanoyl” or “aroyl,” as used herein,alone or in combination, refers to an acyl radical derived from anaryl-substituted alkanecarboxylic acid such as benzoyl, naphthoyl,phenylacetyl, 3-phenylpropionyl(hydrocinnamoyl), 4-phenylbutyryl,(2-naphthyl)acetyl, 4-chlorohydrocinnamoyl, and the like.

The term aryloxy as used herein, alone or in combination, refers to anaryl group attached to the parent molecular moiety through an oxy.

The terms “benzo” and “benz,” as used herein, alone or in combination,refer to the divalent radical C₆H₄═ derived from benzene. Examplesinclude benzothiophene and benzimidazole.

The term “carbamate,” as used herein, alone or in combination, refers toan ester of carbamic acid (—NHCOO—) which may be attached to the parentmolecular moiety from either the nitrogen or acid end, and which may beoptionally substituted as defined herein.

The term “O-carbamyl” as used herein, alone or in combination, refers toa —OC(O)NRR′, group-with R and R′ as defined herein.

The term “N-carbamyl” as used herein, alone or in combination, refers toa ROC(O)NR′— group, with R and R′ as defined herein.

The term “carbonyl,” as used herein, when alone includes formyl [—C(O)H]and in combination is a —C(O)— group.

The term “carboxyl” or “carboxyl,” as used herein, refers to —C(O)OH,O-carboxy, C-carboxy, or the corresponding “carboxylate” anion, such asis in a carboxylic acid salt. An “O-carboxy” group refers to a RC(O)O—group, where R is as defined herein. A “C-carboxy” group refers to a—C(O)OR groups where R is as defined herein.

The term “cyano,” as used herein, alone or in combination, refers to—CN.

The term “cycloalkyl,” or, alternatively, “carbocycle,” as used herein,alone or in combination, refers to a saturated or partially saturatedmonocyclic, bicyclic or tricyclic alkyl radical wherein each cyclicmoiety contains from 3 to 12 carbon atom ring members and which mayoptionally be a benzo fused ring system which is optionally substitutedas defined herein. In certain embodiments, said cycloalkyl will comprisefrom 5 to 7 carbon atoms. Examples of such cycloalkyl radicals includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,octahydronaphthyl, 2,3-dihydro-1H-indenyl, adamantyl and the like.“Bicyclic” and “tricyclic” as used herein are intended to include bothfused ring systems, such as decahydronaphthalene, octahydronaphthaleneas well as the multicyclic (multicentered) saturated or partiallyunsaturated type. The latter type of isomer is exemplified in generalby, bicyclo[1,1,1]pentane, camphor, adamantane, andbicyclo[3,2,1]octane.

The term “ester,” as used herein, alone or in combination, refers to acarboxyl group bridging two moieties linked at carbon atoms.

The term “ether,” as used herein, alone or in combination, typicallyrefers to an oxy group bridging two moieties linked at carbon atoms.“Ether” may also include polyethers, such as, for example,—RO(CH₂)₂O(CH₂)₂O(CH₂)₂OR′, —RO(CH₂)₂O(CH₂)₂OR′, —RO(CH₂)₂OR′, and—RO(CH₂)₂OH.

The term “halo,” or “halogen,” as used herein, alone or in combination,refers to fluorine, chlorine, bromine, or iodine.

The term “haloalkoxy,” as used herein, alone or in combination, refersto a haloalkyl group attached to the parent molecular moiety through anoxygen atom.

The term “haloalkyl,” as used herein, alone or in combination, refers toan alkyl radical having the meaning as defined above wherein one or morehydrogens are replaced with a halogen. Specifically embraced aremonohaloalkyl, dihaloalkyl and polyhaloalkyl radicals. A monohaloalkylradical, for one example, may have an iodo, bromo, chloro or fluoro atomwithin the radical. Dihalo and polyhaloalkyl radicals may have two ormore of the same halo atoms or a combination of different halo radicals.Examples of haloalkyl radicals include fluoromethyl, difluoromethyl,trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl,pentafluoroethyl, heptafluoropropyl, difluorochloromethyl,dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl anddichloropropyl. “Haloalkylene” refers to a haloalkyl group attached attwo or more positions. Examples include fluoromethylene (—CFH—),difluoromethylene (—CF₂—), chloromethylene (—CHCl—) and the like.

The term “heteroalkyl,” as used herein, alone or in combination, refersto a stable straight or branched chain, or cyclic hydrocarbon radical,or combinations thereof, fully saturated or containing from 1 to 3degrees of unsaturation, consisting of the stated number of carbon atomsand from one to three heteroatoms selected from the group consisting ofO, N, and S, and wherein the nitrogen and sulfur atoms may optionally beoxidized and the nitrogen heteroatom may optionally be quaternized. Theheteroatom(s) O, N and S may be placed at any interior position of theheteroalkyl group. Up to two heteroatoms may be consecutive, such as,for example, —CH₂—NH—OCH₃. The term heteroalkyl may include ethers.

The term “heteroaryl,” as used herein, alone or in combination, refersto 3 to 7 membered unsaturated heteromonocyclic rings, or fusedpolycyclic rings in which at least one of the fused rings isunsaturated, wherein at least one atom is selected from the groupconsisting of O, S, and N. In certain embodiments, said heteroaryl willcomprise from 5 to 7 carbon atoms. The term also embraces fusedpolycyclic groups wherein heterocyclic radicals are fused with arylradicals, wherein heteroaryl radicals are fused with other heteroarylradicals, or wherein heteroaryl radicals are fused with cycloalkylradicals. Examples of heteroaryl groups include pyrrolyl, pyrrolinyl,imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl,triazolyl, pyranyl, furyl, thienyl, oxazolyl, isoxazolyl, oxadiazolyl,thiazolyl, thiadiazolyl, isothiazolyl, indolyl, isoindolyl, indolizinyl,benzimidazolyl, quinolyl, isoquinolyl, quinoxalinyl, quinazolinyl,indazolyl, benzotriazolyl, benzodioxolyl, benzopyranyl, benzoxazolyl,benzoxadiazolyl, benzothiazolyl, benzothiadiazolyl, benzofuryl,benzothienyl, chromonyl, coumarinyl, benzopyranyl, tetrahydroquinolinyl,tetrazolopyridazinyl, tetrahydroisoquinolinyl, thienopyridinyl,furopyridinyl, pyrrolopyridinyl and the like. Exemplary tricyclicheterocyclic groups include carbazolyl, benzidolyl, phenanthrolinyl,dibenzofuranyl, acridinyl, phenanthridinyl, xanthenyl and the like.

The terms “heterocycloalkyl” and, interchangeably, “heterocycle,” asused herein, alone or in combination, each refer to a saturated,partially unsaturated, or fully unsaturated monocyclic, bicyclic, ortricyclic heterocyclic radical containing at least one heteroatom asring members, wherein each said heteroatom may be independently selectedfrom the group consisting of nitrogen, oxygen, and sulfur In certainembodiments, said heterocycloalkyl will comprise from 1 to 4 heteroatomsas ring members. In further embodiments, said heterocycloalkyl willcomprise from 1 to 2 heteroatoms ring members. In certain embodiments,said heterocycloalkyl will comprise from 3 to 8 ring members in eachring. In further embodiments, said heterocycloalkyl will comprise from 3to 7 ring members in each ring. In yet further embodiments, saidheterocycloalkyl will comprise from 5 to 6 ring members in each ring.“Heterocycloalkyl” and “heterocycle” are intended to include sugars,sulfones, sulfoxides, N-oxides of tertiary nitrogen ring members, andcarbocyclic fused and benzo fused ring systems; additionally, both termsalso include systems where a heterocycle ring is fused to an aryl group,as defined herein, or an additional heterocycle group. Examples ofheterocycloalkyl groups include aziridinyl, azetidinyl,1,3-benzodioxolyl, dihydroisoindolyl, dihydroisoquinolinyl,dihydrocinnolinyl, dihydrobenzodioxinyl,dihydro[1,3]oxazolo[4,5-b]pyridinyl, benzothiazolyl, dihydroindolyl,dihy-dropyridinyl, 1,3-dioxanyl, 1,4-dioxanyl, 1,3-dioxolanyl,isoindolinyl, morpholinyl, piperazinyl, pyrrolidinyl,tetrahydropyridinyl, piperidinyl, thiomorpholinyl, and the like. Theheterocycloalkyl groups may be optionally substituted unlessspecifically prohibited.

The term “hydrazinyl” as used herein, alone or in combination, refers totwo amino groups joined by a single bond, i.e., —N—N—.

The term “hydroxamic acid” as used herein, refers to —C(O)ON(R)O(R′),wherein R and R′ are as defined herein, or the corresponding“hydroxamate” anion, including any corresponding hydroxamic acid salt.

The term “hydroxy,” as used herein, alone or in combination, refers to—OH.

The term “hydroxyalkyl,” as used herein, alone or in combination, refersto a hydroxy group attached to the parent molecular moiety through analkyl group.

The term “imino,” as used herein, alone or in combination, refers to═N—.

The term “iminohydroxy,” as used herein, alone or in combination, refersto ═N(OH) and ═N—O—.

The term “isocyanato” refers to a —NCO group.

The term “isothiocyanato” refers to a —NCS group.

The phrase “linear chain of atoms” refers to the longest straight chainof atoms independently selected from carbon, nitrogen, oxygen andsulfur.

The term “lower,” as used herein, alone or in combination, meanscontaining from 1 to and including 6 carbon atoms.

The term “mercaptyl” as used herein, alone or in combination, refers toan RS— group, where R is as defined herein.

The term “nitro,” as used herein, alone or in combination, refers to—NO₂.

The terms “oxy” or “oxa” as used herein, alone or in combination, referto —O—.

The term “oxo,” as used herein, alone or in combination, refers to ═O.

The term “perhaloalkoxy” refers to an alkoxy group where all of thehydrogen atoms are replaced by halogen atoms.

The term “perhaloalkyl” as used herein, alone or in combination, refersto an alkyl group where all of the hydrogen atoms are replaced byhalogen atoms.

The term “phosphoamide” as used herein, alone or in combination, refersto a phosphate group [(OH)₂P(O)O—] in which one or more of the hydroxylgroups has been replaced by nitrogen, amino, or amido.

The term “phosphonate” as used herein, alone or in combination, refersto a group of the form ROP(OR′)(OR)O— wherein R and R′ are selected fromthe group consisting of hydrogen, alkyl, acyl, heteroalkyl, aryl,cycloalkyl, heteroaryl, and heterocycloalkyl, any of which maythemselves be optionally substituted. “Phosphonate” includes “phosphate[(OH)₂P(O)O—] and related phosphoric acid anions which may form salts.

The terms “sulfonate,” “sulfonic acid,” and “sulfonic,” as used herein,alone or in combination, refers to the —SO₃H group and its anion as thesulfonic acid is used in salt formation.

The term “sulfanyl,” as used herein, alone or in combination, refers to—S—.

The term “sulfinyl,” as used herein, alone or in combination, refers to—S(O)—.

The term “sulfonyl,” as used herein, alone or in combination, refers to—S(O)₂—.

The term “N-sulfonamido” refers to a RS(═O)₂NR′— group with R and R′ asdefined herein.

The term “S-sulfonamido” refers to a —S(═O)₂NRR′, group, with R and R′as defined herein.

The terms “thia” and “thio,” as used herein, alone or in combination,refer to a —S— group or an ether wherein the oxygen is replaced withsulfur. The oxidized derivatives of the thio group, namely sulfinyl andsulfonyl, are included in the definition of thia and thio.

The term “thiol,” as used herein, alone or in combination, refers to an—SH group.

The term “thiocarbonyl,” as used herein, when alone includes thioformyl—C(S)H and in combination is a —C(S)— group.

The term “N-thiocarbamyl” refers to an ROC(S)NR′— group, with R and R′as defined herein.

The term “O-thiocarbamyl” refers to a —OC(S)NRR′, group with R and R′ asdefined herein.

The term “thiocyanato” refers to a —CNS group.

The term “trihalomethanesulfonamido” refers to a X₃CS(O)₂NR— group withX is a halogen and R as defined herein.

The term “trihalomethanesulfonyl” refers to a X₃CS(O)₂— group where X isa halogen.

The term “trihalomethoxy” refers to a X₃CO— group where X is a halogen.

The term “trisubstituted silyl,” as used herein, alone or incombination, refers to a silicone group substituted at its three freevalences with groups as listed herein under the definition ofsubstituted amino. Examples include trimethysilyl,tert-butyldimethylsilyl, triphenylsilyl and the like.

Any definition herein may be used in combination with any otherdefinition to describe a composite structural group. By convention, thetrailing element of any such definition is that which attaches to theparent moiety. For example, the composite group alkylamido wouldrepresent an alkyl group attached to the parent molecule through anamido group, and the term alkoxyalkyl would represent an alkoxy groupattached to the parent molecule through an alkyl group.

When a group is defined to be “null,” what is meant is that said groupis absent. A “null” group occurring between two other groups may also beunderstood to be a collapsing of flanking groups. For example, if in—(CH₂)_(s)G¹G²G³, the element G² were null, said group would become—(CH₂)_(s)G¹G³.

The term “optionally substituted” means the anteceding group may besubstituted or unsubstituted. When substituted, the substituents of an“optionally substituted” group may include, without limitation, one ormore substituents independently selected from the following groups or aparticular designated set of groups, alone or in combination: loweralkyl, lower alkenyl, lower alkynyl, lower alkanoyl, lower heteroalkyl,lower heterocycloalkyl, lower haloalkyl, lower haloalkenyl, lowerhaloalkynyl, lower perhaloalkyl, lower perhaloalkoxy, lower cycloalkyl,phenyl, aryl, aryloxy, lower alkoxy, lower haloalkoxy, oxo, loweracyloxy, carbonyl, carboxyl, lower alkylcarbonyl, lower carboxyester,lower carboxamido, cyano, hydrogen, halogen, hydroxy, amino, loweralkylamino, arylamino, amido, nitro, thiol, lower alkylthio, lowerhaloalkylthio, lower perhaloalkylthio, arylthio, sulfonate, sulfonicacid, trisubstituted silyl, N₃, SH, SCH₃, C(O)CH₃, CO₂CH₃, CO₂H,pyridinyl, thiophene, furanyl, lower carbamate, and lower urea. Twosubstituents may be joined together to form a fused five-, six-, orseven-membered carbocyclic or heterocyclic ring consisting of zero tothree heteroatoms, for example forming methylenedioxy or ethylenedioxy.An optionally substituted group may be unsubstituted (e.g., —CH₂CH₃),fully substituted (e.g., —CF₂CF₃), monosubstituted (e.g., —CH₂CH₂F) orsubstituted at a level anywhere in-between fully substituted andmonosubstituted (e.g., —CH₂CF₃). Where substituents are recited withoutqualification as to substitution, both substituted and unsubstitutedforms are encompassed. Where a substituent is qualified as“substituted,” the substituted form is specifically intended.Additionally, different sets of optional substituents to a particularmoiety may be defined as needed; in these cases, the optionalsubstitution will be as defined, often immediately following the phrase,“optionally substituted with.”

The term R or the term R′, appearing by itself and without a numberdesignation, unless otherwise defined, refers to a moiety selected fromthe group consisting of hydrogen, hydroxyl, halogen, alkyl, cycloalkyl,heteroalkyl, aryl, heteroaryl and heterocycloalkyl, any of which may beoptionally substituted. Such R and R′ groups should be understood to beoptionally substituted as defined herein. Whether an R group has anumber designation or not, every R group, including R, R′ and R″ wheren=(1, 2, 3, . . . n), every substituent, and every term should beunderstood to be independent of every other in terms of selection from agroup. Should any variable, substituent, or term (e.g. aryl,heterocycle, R, etc.) occur more than one time in a formula or genericstructure, its definition at each occurrence is independent of thedefinition at every other occurrence. Those of skill in the art willfurther recognize that certain groups may be attached to a parentmolecule or may occupy a position in a chain of elements from either endas written. Thus, by way of example only, an unsymmetrical group such as—C(O)N(R)— may be attached to the parent moiety at either the carbon orthe nitrogen.

Asymmetric centers exist in the compounds of the present invention.These centers are designated by the symbols “R” or “S,” depending on theconfiguration of substituents around the chiral carbon atom. It shouldbe understood that the invention encompasses all stereochemical isomericforms, including diastereomeric, enantiomeric, and epimeric forms, aswell as d-isomers and 1-isomers, and mixtures thereof. Individualstereoisomers of compounds can be prepared synthetically fromcommercially available starting materials which contain chiral centersor by preparation of mixtures of enantiomeric products followed byseparation such as conversion to a mixture of diastereomers followed byseparation or recrystallization, chromatographic techniques, directseparation of enantiomers on chiral chromatographic columns, or anyother appropriate method known in the art. Starting compounds ofparticular stereochemistry are either commercially available or can bemade and resolved by techniques known in the art. Additionally, thecompounds of the present invention may exist as geometric isomers. Thepresent invention includes all cis, trans, syn, anti, entgegen (E), andzusammen (Z) isomers as well as the appropriate mixtures thereof.Additionally, compounds may exist as tautomers, including keto-enoltautomers; all tautomeric isomers are provided by this invention.Additionally, the compounds of the present invention can exist inunsolvated as well as solvated forms with pharmaceutically acceptablesolvents such as water, ethanol, and the like. In general, the solvatedforms are considered equivalent to the unsolvated forms for the purposesof the present invention.

The term “bond” refers to a covalent linkage between two atoms, or twomoieties when the atoms joined by the bond are considered to be part oflarger substructure. A bond may be single, double, or triple unlessotherwise specified. A dashed line between two atoms in a drawing of amolecule indicates that an additional bond may be present or absent atthat position.

The term “disease” as used herein is intended to be generallysynonymous, and is used interchangeably with, the terms “disorder” and“condition” (as in medical condition), in that all reflect an abnormalcondition of the body or of one of its parts that impairs normalfunctioning and is typically manifested by distinguishing signs andsymptoms.

The term “combination therapy” means the administration of two or moretherapeutic agents to treat a therapeutic condition or disorderdescribed in the present disclosure. Such administration encompassesco-administration of these therapeutic agents in a substantiallysimultaneous manner, such as in a single capsule having a fixed ratio ofactive ingredients or in multiple, separate capsules for each activeingredient. In addition, such administration also encompasses use ofeach type of therapeutic agent in a sequential manner. In either case,the treatment regimen will provide beneficial effects of the drugcombination in treating the conditions or disorders described herein.

“Rho kinase inhibitor” is used herein to refer to a compound thatexhibits an IC₅₀ with respect to Rho kinase activity of no more thanabout 100 μM and more typically not more than about 50 μM, as measuredin the Rho kinase assay described generally hereinbelow. “IC₅₀” is thatconcentration of inhibitor which reduces the activity of an enzyme(e.g., Rho kinase) to half-maximal level. Certain representativecompounds of the present invention have been discovered to exhibitinhibition against Rho kinase. In certain embodiments, compounds willexhibit an IC₅₀ with respect to Rho kinase of no more than about 10 μM;in further embodiments, compounds will exhibit an IC₅₀ with respect toRho kinase of no more than about 5 μM; in yet further embodiments,compounds will exhibit an IC₅₀ with respect to Rho kinase of not morethan about 1 μM, as measured in the Rho kinase assay described herein.In yet further embodiments, compounds will exhibit an IC₅₀ with respectto Rho kinase of not more than about 200 nM.

The phrase “therapeutically effective” is intended to qualify the amountof active ingredients used in the treatment of a disease or disorder.This amount will achieve the goal of reducing or eliminating the saiddisease or disorder.

As used herein, reference to “treatment” of a patient is intended toinclude prophylaxis. The term “patient” means all mammals includinghumans. Examples of patients include humans, cows, dogs, cats, goats,sheep, pigs, and rabbits. Preferably, the patient is a human.

The term “prodrug” refers to a compound that is made more active invivo. Certain of the present compounds can also exist as prodrugs, asdescribed in Hydrolysis in Drug and Prodrug Metabolism: Chemistry,Biochemistry, and Enzymology (Testa, Bernard and Mayer, Joachim M.Wiley-VHCA, Zurich, Switzerland 2003). Prodrugs of the compoundsdescribed herein are structurally modified forms of the compound thatreadily undergo chemical changes under physiological conditions toprovide the compound. Additionally, prodrugs can be converted to thecompound by chemical or biochemical methods in an ex vivo environment.For example, prodrugs can be slowly converted to a compound when placedin a transdermal patch reservoir with a suitable enzyme or chemicalreagent. Prodrugs are often useful because, in some situations, they maybe easier to administer than the compound, or parent drug. They may, forinstance, be bioavailable by oral administration whereas the parent drugis not. The prodrug may also have improved solubility in pharmaceuticalcompositions over the parent drug. A wide variety of prodrug derivativesare known in the art, such as those that rely on hydrolytic cleavage oroxidative activation of the prodrug. An example, without limitation, ofa prodrug would be a compound which is administered as an ester (the“prodrug”), but then is metabolically hydrolyzed to the carboxylic acid,the active entity. Additional examples include peptidyl derivatives of acompound. The term “therapeutically acceptable prodrug,” refers to thoseprodrugs or zwitterions which are suitable for use in contact with thetissues of patients without undue toxicity, irritation, and allergicresponse, are commensurate with a reasonable benefit/risk ratio, and areeffective for their intended use.

The compounds of the present invention can exist as therapeuticallyacceptable salts. The present invention includes compounds listed abovein the form of salts, including acid addition salts. Suitable saltsinclude those formed with both organic and inorganic acids. Such acidaddition salts will normally be pharmaceutically acceptable. However,salts of non-pharmaceutically acceptable salts may be of utility in thepreparation and purification of the compound in question. Basic additionsalts may also be formed and be pharmaceutically acceptable. For a morecomplete discussion of the preparation and selection of salts, refer toPharmaceutical Salts: Properties, Selection, and Use (Stahl, P.Heinrich. Wiley-VCHA, Zurich, Switzerland, 2002).

The term “therapeutically acceptable salt,” as used herein, representssalts or zwitterionic forms of the compounds of the present inventionwhich are water or oil-soluble or dispersible and therapeuticallyacceptable as defined herein. The salts can be prepared during the finalisolation and purification of the compounds or separately by reactingthe appropriate compound in the form of the free base with a suitableacid. Representative acid addition salts include acetate, adipate,alginate, L-ascorbate, aspartate, benzoate, benzenesulfonate(besylate),bisulfate, butyrate, camphorate, camphorsulfonate, citrate, digluconate,formate, fumarate, gentisate, glutarate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethansulfonate(isethionate),lactate, maleate, malonate, DL-mandelate, mesitylenesulfonate,methanesulfonate, naphthylenesulfonate, nicotinate,2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate,3-phenylproprionate, phosphonate, picrate, pivalate, propionate,pyroglutamate, succinate, sulfonate, tartrate, L-tartrate,trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate,para-toluenesulfonate(p-tosylate), and undecanoate. Also, basic groupsin the compounds of the present invention can be quaternized withmethyl, ethyl, propyl, and butyl chlorides, bromides, and iodides;dimethyl, diethyl, dibutyl, and diamyl sulfates; decyl, lauryl,myristyl, and steryl chlorides, bromides, and iodides; and benzyl andphenethyl bromides. Examples of acids which can be employed to formtherapeutically acceptable addition salts include inorganic acids suchas hydrochloric, hydrobromic, sulfuric, and phosphoric, and organicacids such as oxalic, maleic, succinic, and citric. Salts can also beformed by coordination of the compounds with an alkali metal or alkalineearth ion. Hence, the present invention contemplates sodium, potassium,magnesium, and calcium salts of the compounds disclosed herein, and thelike.

Basic addition salts can be prepared during the final isolation andpurification of the compounds by reacting a carboxyl group with asuitable base such as the hydroxide, carbonate, or bicarbonate of ametal cation or with ammonia or an organic primary, secondary, ortertiary amine. The cations of therapeutically acceptable salts includelithium, sodium, potassium, calcium, magnesium, and aluminum, as well asnontoxic quaternary amine cations such as ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, diethylamine, ethylamine, tributylamine, pyridine,N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine,dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine,1-ephenamine, and N,N′-dibenzylethylenediamine. Other representativeorganic amines useful for the formation of base addition salts includeethylenediamine, ethanolamine, diethanolamine, piperidine, andpiperazine.

While it may be possible for the compounds of the subject invention tobe administered as the raw chemical, it is also possible to present themas a pharmaceutical formulation. Accordingly, provided herein arepharmaceutical formulations which comprise one or more of certaincompounds of the present invention, or one or more pharmaceuticallyacceptable salts, esters, prodrugs, amides, or solvates thereof,together with one or more pharmaceutically acceptable carriers thereofand optionally one or more other therapeutic ingredients. The carrier(s)must be “acceptable” in the sense of being compatible with the otheringredients of the formulation and not deleterious to the recipientthereof. Proper formulation is dependent upon the route ofadministration chosen. Any of the well-known techniques, carriers, andexcipients may be used as suitable and as understood in the art; e.g.,in Remington's Pharmaceutical Sciences. The pharmaceutical compositionsdisclosed herein may be manufactured in any manner known in the art,e.g., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping orcompression processes.

The formulations include those suitable for oral, parenteral (includingsubcutaneous, intradermal, intramuscular, intravenous, intraarticular,and intramedullary), intraperitoneal, transmucosal, transdermal, rectaland topical (including dermal, buccal, sublingual and intraocular)administration although the most suitable route may depend upon forexample the condition and disorder of the recipient. The formulationsmay conveniently be presented in unit dosage form and may be prepared byany of the methods well known in the art of pharmacy. Typically, thesemethods include the step of bringing into association a compound of thesubject invention or a pharmaceutically acceptable salt, ester, amide,prodrug or solvate thereof (“active ingredient”) with the carrier whichconstitutes one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation the active ingredient with liquid carriers or finely dividedsolid carriers or both and then, if necessary, shaping the product intothe desired formulation.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste.

Pharmaceutical preparations which can be used orally include tablets,push-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. Tablets maybe made by compression or molding, optionally with one or more accessoryingredients. Compressed tablets may be prepared by compressing in asuitable machine the active ingredient in a free-flowing form such as apowder or granules, optionally mixed with binders, inert diluents, orlubricating, surface active or dispersing agents. Molded tablets may bemade by molding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets may optionally becoated or scored and may be formulated so as to provide slow orcontrolled release of the active ingredient therein. All formulationsfor oral administration should be in dosages suitable for suchadministration. The push-fit capsules can contain the active ingredientsin admixture with filler such as lactose, binders such as starches,and/or lubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active compounds may be dissolved orsuspended in suitable liquids, such as fatty oils, liquid paraffin, orliquid polyethylene glycols. In addition, stabilizers may be added.Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. The formulations may be presentedin unit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in powder form or in a freeze-dried(lyophilized) condition requiring only the addition of the sterileliquid carrier, for example, saline or sterile pyrogen-free water,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

Formulations for parenteral administration include aqueous andnon-aqueous (oily) sterile injection solutions of the active compoundswhich may contain antioxidants, buffers, bacteriostats and solutes whichrender the formulation isotonic with the blood of the intendedrecipient; and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents and thickening agents. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Aqueous injection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

For buccal or sublingual administration, the compositions may take theform of tablets, lozenges, pastilles, or gels formulated in conventionalmanner. Such compositions may comprise the active ingredient in aflavored basis such as sucrose and acacia or tragacanth.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter, polyethylene glycol, or otherglycerides.

Certain compounds of the present invention may be administeredtopically, that is by non-systemic administration. This includes theapplication of a compound of the present invention externally to theepidermis or the buccal cavity and the instillation of such a compoundinto the ear, eye and nose, such that the compound does notsignificantly enter the blood stream. In contrast, systemicadministration refers to oral, intravenous, intraperitoneal andintramuscular administration.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin tothe site of inflammation such as gels, liniments, lotions, creams,ointments or pastes, and drops suitable for administration to the eye,ear or nose. The active ingredient for topical administration maycomprise, for example, from 0.001% to 10% w/w (by weight) of theformulation. In certain embodiments, the active ingredient may compriseas much as 10% w/w. In other embodiments, it may comprise less than 5%w/w. In certain embodiments, the active ingredient may comprise from 2%w/w to 5% w/w. In other embodiments, it may comprise from 0.1% to 1% w/wof the formulation.

Gels for topical or transdermal administration may comprise, generally,a mixture of volatile solvents, nonvolatile solvents, and water. Incertain embodiments, the volatile solvent component of the bufferedsolvent system may include lower (C1-C6)alkyl alcohols, lower alkylglycols and lower glycol polymers. In further embodiments, the volatilesolvent is ethanol. The volatile solvent component is thought to act asa penetration enhancer, while also producing a cooling effect on theskin as it evaporates. The nonvolatile solvent portion of the bufferedsolvent system is selected from lower alkylene glycols and lower glycolpolymers. In certain embodiments, propylene glycol is used. Thenonvolatile solvent slows the evaporation of the volatile solvent andreduces the vapor pressure of the buffered solvent system. The amount ofthis nonvolatile solvent component, as with the volatile solvent, isdetermined by the pharmaceutical compound or drug being used. When toolittle of the nonvolatile solvent is in the system, the pharmaceuticalcompound may crystallize due to evaporation of volatile solvent, whilean excess may result in a lack of bioavailability due to poor release ofdrug from solvent mixture. The buffer component of the buffered solventsystem may be selected from any buffer commonly used in the art; incertain embodiments, water is used. A common ratio of ingredients isabout 20% of the nonvolatile solvent, about 40% of the volatile solvent,and about 40% water. There are several optional ingredients which can beadded to the topical composition. These include, but are not limited to,chelators and gelling agents. Appropriate gelling agents can include,but are not limited to, semisynthetic cellulose derivatives (such ashydroxypropylmethylcellulose) and synthetic polymers, and cosmeticagents.

Lotions include those suitable for application to the skin or eye. Aneye lotion may comprise a sterile aqueous solution optionally containinga bactericide and may be prepared by methods similar to those for thepreparation of drops. Lotions or liniments for application to the skinmay also include an agent to hasten drying and to cool the skin, such asan alcohol or acetone, and/or a moisturizer such as glycerol or an oilsuch as castor oil or arachis oil.

Creams, ointments or pastes are semi-solid formulations of the activeingredient for external application. They may be made by mixing theactive ingredient in finely-divided or powdered form, alone or insolution or suspension in an aqueous or non-aqueous fluid, with the aidof suitable machinery, with a greasy or non-greasy base. The base maycomprise hydrocarbons such as hard, soft or liquid paraffin, glycerol,beeswax, a metallic soap; a mucilage; an oil of natural origin such asalmond, corn, arachis, castor or olive oil; wool fat or its derivativesor a fatty acid such as stearic or oleic acid together with an alcoholsuch as propylene glycol or a macrogel. The formulation may incorporateany suitable surface active agent such as an anionic, cationic ornon-ionic surfactant such as a sorbitan ester or a polyoxyethylenederivative thereof. Suspending agents such as natural gums, cellulosederivatives or inorganic materials such as silicaceous silicas, andother ingredients such as lanolin, may also be included.

Drops may comprise sterile aqueous or oily solutions or suspensions andmay be prepared by dissolving the active ingredient in a suitableaqueous solution of a bactericidal and/or fungicidal agent and/or anyother suitable preservative, and, in certain embodiments, including asurface active agent. The resulting solution may then be clarified byfiltration, transferred to a suitable container which is then sealed andsterilized by autoclaving or maintaining at 98-100° C. for half an hour.Alternatively, the solution may be sterilized by filtration andtransferred to the container by an aseptic technique. Examples ofbactericidal and fungicidal agents suitable for inclusion in the dropsare phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride(0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for thepreparation of an oily solution include glycerol, diluted alcohol andpropylene glycol.

Formulations for topical administration in the mouth, for examplebuccally or sublingually, include lozenges comprising the activeingredient in a flavored basis such as sucrose and acacia or tragacanth,and pastilles comprising the active ingredient in a basis such asgelatin and glycerin or sucrose and acacia.

For administration by inhalation, compounds may be convenientlydelivered from an insufflator, nebulizer pressurized packs or otherconvenient means of delivering an aerosol spray. Pressurized packs maycomprise a suitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Alternatively, for administration by inhalation or insufflation, thecompounds according to the invention may take the form of a dry powdercomposition, for example a powder mix of the compound and a suitablepowder base such as lactose or starch. The powder composition may bepresented in unit dosage form, in for example, capsules, cartridges,gelatin or blister packs from which the powder may be administered withthe aid of an inhalator or insufflator.

Preferred unit dosage formulations are those containing an effectivedose, as herein below recited, or an appropriate fraction thereof, ofthe active ingredient.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations described above may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavoring agents.

Compounds may be administered orally or via injection at a dose of from0.1 to 500 mg/kg per day. The dose range for adult humans is generallyfrom 5 mg to 2 g/day. Tablets or other forms of presentation provided indiscrete units may conveniently contain an amount of one or morecompounds which is effective at such dosage or as a multiple of thesame, for instance, units containing 5 mg to 500 mg, usually around 10mg to 200 mg.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration.

The compounds can be administered in various modes, e.g. orally,topically, or by injection. The precise amount of compound administeredto a patient will be the responsibility of the attendant physician. Thespecific dose level for any particular patient will depend upon avariety of factors including the activity of the specific compoundemployed, the age, body weight, general health, sex, diets, time ofadministration, route of administration, rate of excretion, drugcombination, the precise disorder being treated, and the severity of theindication or condition being treated. Also, the route of administrationmay vary depending on the condition and its severity.

In certain instances, it may be appropriate to administer at least oneof the compounds described herein (or a pharmaceutically acceptablesalt, ester, or prodrug thereof) in combination with another therapeuticagent. By way of example only, if one of the side effects experienced bya patient upon receiving one of the compounds herein is hypertension,then it may be appropriate to administer an anti-hypertensive agent incombination with the initial therapeutic agent. Or, by way of exampleonly, the therapeutic effectiveness of one of the compounds describedherein may be enhanced by administration of an adjuvant (i.e., by itselfthe adjuvant may only have minimal therapeutic benefit, but incombination with another therapeutic agent, the overall therapeuticbenefit to the patient is enhanced). Or, by way of example only, thebenefit of experienced by a patient may be increased by administeringone of the compounds described herein with another therapeutic agent(which also includes a therapeutic regimen) that also has therapeuticbenefit. By way of example only, in a treatment for diabetes involvingadministration of one of the compounds described herein, increasedtherapeutic benefit may result by also providing the patient withanother therapeutic agent for diabetes. In any case, regardless of thedisease, disorder or condition being treated, the overall benefitexperienced by the patient may simply be additive of the two therapeuticagents or the patient may experience a synergistic benefit.

In any case, the multiple therapeutic agents (at least one of which is acompound of the present invention) may be administered in any order oreven simultaneously. If simultaneously, the multiple therapeutic agentsmay be provided in a single, unified form, or in multiple forms (by wayof example only, either as a single pill or as two separate pills). Oneof the therapeutic agents may be given in multiple doses, or both may begiven as multiple doses. If not simultaneous, the timing between themultiple doses may be any duration of time ranging from a few minutes tofour weeks.

Thus, in another aspect, the present invention provides methods fortreating Rho kinase-mediated disorders in a human or animal subject inneed of such treatment comprising administering to said subject anamount of a compound of the present invention effective to reduce orprevent said disorder in the subject in combination with at least oneadditional agent for the treatment of said disorder that is known in theart. In a related aspect, the present invention provides therapeuticcompositions comprising at least one compound of the present inventionin combination with one or more additional agents for the treatment ofRho kinase-mediated disorders.

Compounds of the subject invention may be useful in treating Rhokinase-mediated disease, disorders and conditions. In certainembodiments, said compounds may find use in treating acute and chronicpain and inflammation. The compounds of the present invention may beuseful to treat patients with neuropathy, neuropathic pain, orinflammatory pain such as reflex sympathetic dystrophy/causalgia (nerveinjury), peripheral neuropathy (including diabetic neuropathy),intractable cancer pain, complex regional pain syndrome, and entrapmentneuropathy (carpel tunnel syndrome). The compounds may also be useful inthe treatment of pain associated with acute herpes zoster (shingles),postherpetic neuralgia (PHN), and associated pain syndromes such asocular pain. The compounds may further be useful as analgesics in thetreatment of pain such as surgical analgesia, or as an antipyretic forthe treatment of fever. Pain indications include, but are not limitedto, post-surgical pain for various surgical procedures includingpost-cardiac surgery, dental pain/dental extraction, pain resulting fromcancer, muscular pain, mastalgia, pain resulting from dermal injuries,lower back pain, headaches of various etiologies, including migraine,and the like. The compounds may also be useful for the treatment ofpain-related disorders such as tactile allodynia and hyperalgesia. Thepain may be somatogenic (either nociceptive or neuropathic), acuteand/or chronic. The Rho kinase inhibitors of the subject invention mayalso be useful in conditions where NSAIDs, morphine or fentanyl opiatesand/or other opioid analgesics would traditionally be administered.

Furthermore, compounds of the subject invention may be used in thetreatment or prevention of opiate tolerance in patients needingprotracted opiate analgesics, and benzodiazepine tolerance in patientstaking benzodiazepines, and other addictive behavior, for example,nicotine addiction, alcoholism, and eating disorders. Moreover, thecompounds and methods of the present invention may be useful in thetreatment or prevention of drug withdrawal symptoms, for exampletreatment or prevention of symptoms of withdrawal from opiate, alcohol,or tobacco addiction.

In addition, compounds of the subject invention may be used to treatinsulin resistance and other metabolic disorders such as atherosclerosisthat are typically associated with an exaggerated inflammatorysignaling.

The present invention encompasses therapeutic methods using novelselective Rho kinase inhibitors to treat or prevent respiratory diseaseor conditions, including therapeutic methods of use in medicine forpreventing and treating a respiratory disease or condition including:asthmatic conditions including allergen-induced asthma, exercise-inducedasthma, pollution-induced asthma, cold-induced asthma, andviral-induced-asthma; asthma-related diseases such as airwayhyperreactivity and small airway disease; chronic obstructive pulmonarydiseases including chronic bronchitis with normal airflow, chronicbronchitis with airway obstruction (chronic obstructive bronchitis),emphysema, asthmatic bronchitis, and bullous disease; and otherpulmonary diseases involving inflammation including bronchiolitis,bronchioectasis, cystic fibrosis, pigeon fancier's disease, farmer'slung, acute respiratory distress syndrome, pneumonia, pneumonitis,aspiration or inhalation injury, fat embolism in the lung, acidosisinflammation of the lung, acute pulmonary edema, acute mountainsickness, acute pulmonary hypertension, persistent pulmonaryhypertension of the newborn, perinatal aspiration syndrome, hyalinemembrane disease, acute pulmonary thromboembolism, heparin-protaminereactions, sepsis, status asthamticus, hypoxia, dyspnea, hypercapnea,hyperinflation, hypoxemia, and cough. Further, compounds disclosedherein would find use in the treatment of allergic disorders such asdelayed type hypersensitivity reaction, allergic contact dermatitis,allergic rhinitis, and chronic sinusitis.

Other disorders or conditions which may be treated by the compounds ofthe present invention include inflammation and related disorders. Thecompounds of the present invention may be useful as anti-inflammatoryagents with the additional benefit of having significantly less harmfulside effects. The compounds may be useful to treat arthritis, includingbut not limited to rheumatoid arthritis, spondyloarthropathies, goutyarthritis, osteoarthritis, juvenile arthritis, acute rheumaticarthritis, enteropathic arthritis, neuropathic arthritis, psoriaticarthritis, reactive arthritis (Reiter's syndrome), and pyogenicarthritis, and autoimmune diseases, including systemic lupuserythematosus, hemolytic syndromes, autoimmune hepatitis, autoimmuneneuropathy, vitiglio (autoimmune thyroiditis), Hashimoto's thyroiditis,anemias, myositis including polymyositis, alopecia greata, Goodpasture'ssyndrome, hypophytis, and pulmonary fibrosis.

The compounds may also be useful in treating osteoporosis and otherrelated bone disorders.

These compounds may also be used to treat gastrointestinal conditionssuch as reflux esophagitis, diarrhea, inflammatory bowel disease,Crohn's disease, gastritis, irritable bowel syndrome, Graves' disease(hyperthyroidism), necrotizing enterocolitis, and ulcerative colitis.The compounds may also be used in the treatment of pulmonaryinflammation, such as that associated with viral infections and cysticfibrosis.

In addition, compounds of invention may also be useful in organtransplant patients either alone or in combination with conventionalimmunomodulators. Examples of conditions to be treated in said patientsinclude graft vs. host reaction (i.e., graft vs. host disease),allograft rejections (e.g., acute allograft rejection, and chronicallograft rejection), transplant reperfusion injury, and earlytransplantation rejection (e.g., acute allograft rejection).

Yet further, the compounds of the invention may be useful in thetreatment of pruritis and vitaligo.

The compounds of the present invention may also be useful in treatingtissue damage in such diseases as vascular diseases, migraine headaches,periarteritis nodosa, thyroiditis, aplastic anemia, Hodgkin's disease,sclerodoma, rheumatic fever, type I diabetes, neuromuscular junctiondisease including myasthenia gravis, white matter disease includingmultiple sclerosis, sarcoidosis, nephritis, nephrotic syndrome,Langerhans' cell histiocytosis, glomerulonephritis, reperfusion injury,pancreatitis, interstitial cystitis, Behcet's syndrome, polymyositis,gingivitis, periodontis, hypersensitivity, swelling occurring afterinjury, ischemias including myocardial ischemia, cardiovascularischemia, and ischemia secondary to cardiac arrest, cirrhosis, septicshock, endotoxic shock, gram negative sepsis, toxic shock syndrome,stroke, ischemia reperfusion injury, multi-organ dysfunction, restenosisincluding restenosis following coronary bypass surgery, and the like.

The compounds of the subject invention may also be useful for thetreatment of certain diseases and disorders of the nervous system.Central nervous system disorders in which Rho kinase inhibition may beuseful include cortical dementias including Alzheimer's disease and mildcognitive impairment (MCI), central nervous system damage resulting fromstroke, ischemias including cerebral ischemia (both focal ischemia,thrombotic stroke and global ischemia (for example, secondary to cardiacarrest), and trauma. Neurodegenerative disorders in which Rho kinaseinhibition may be useful include nerve degeneration or nerve necrosis indisorders such as hypoxia, hypoglycemia, epilepsy, and in cases ofcentral nervous system (CNS) trauma (such as spinal cord and headinjury), hyperbaric oxygen convulsions and toxicity, dementia (e.g.pre-senile dementia), and AIDS-related dementia, cachexia, Sydenham'schorea, Huntington's disease, Parkinson's Disease, amyotrophic lateralsclerosis (ALS), multiple sclerosis, Korsakoff's syndrome, andimbecility relating to a cerebral vessel disorder. Further disorders inwhich Rho kinase inhibition might prove useful include neuropathies ofthe central and peripheral nervous system (including, for example, IgAneuropathy, membranous neuropathy and idiopathic neuropathy), chronicinflammatory demyelinating polyneuropathy, transverse myelitis,Gullain-Barre disease, encephalitis, and cancers of the nervous system.Disorders of CNS function in which Rho kinase inhibitors may find useinclude sleeping disorders, schizophrenia, depression, depression orother symptoms associated with Premenstrual Syndrome (PMS), and anxiety.

Furthermore, the compounds of the present invention may also be usefulin inhibiting Rho kinase activity for the amelioration of systemicdisorders including septic and/or toxic hemorrhagic shock induced by awide variety of agents; as a therapy with cytokines such as TNF, IL-1and IL-2; and as an adjuvant to short term immunosuppression intransplant therapy.

Still other disorders or conditions which may be treated by thecompounds of the subject invention include the prevention or treatmentof cancer, such as colorectal cancer, and cancer of the breast, lung,prostate, bladder, cervix and skin. Compounds of the invention may beused in the treatment and prevention of neoplasias including but notlimited to brain cancer, bone cancer, leukemia, lymphoma, epithelialcell-derived neoplasia (epithelial carcinoma) such as basal cellcarcinoma, adenocarcinoma, gastrointestinal cancer such as lip cancer,mouth cancer, esophageal cancer, small bowel cancer and stomach cancer,colon cancer, liver cancer, bladder cancer, pancreas cancer, ovarycancer, cervical cancer, lung cancer, breast cancer and skin cancer,such as squamous cell and basal cell cancers, prostate cancer, renalcell carcinoma, and other known cancers that effect epithelial cellsthroughout the body. The neoplasia can be selected from gastrointestinalcancer, liver cancer, bladder cancer, pancreas cancer, ovary cancer,prostate cancer, cervical cancer, lung cancer, breast cancer and skincancer, such as squamous cell and basal cell cancers. The presentcompounds and methods may also be used to treat the fibrosis whichoccurs with radiation therapy. The present compounds and methods may beused to treat subjects having adenomatous polyps, including those withfamilial adenomatous polyposis (FAP). Additionally, the presentcompounds and methods may be used to prevent polyps from forming inpatients at risk of FAP.

The compounds of the subject invention may be used in the treatment ofophthalmic diseases, such as dry eye, glaucoma, cornealneovascularization, optic neuritis, Sjogren's syndrome, retinal gangliondegeneration, ocular ischemia, retinitis, retinopathies, uveitis, ocularphotophobia, and of inflammation and pain associated with acute injuryto the eye tissue. Specifically, the compounds may be used to treatglaucomatous retinopathy and/or diabetic retinopathy. The compounds mayalso be used to treat post-operative inflammation or pain as fromophthalmic surgery such as cataract surgery and refractive surgery.

The compounds of the subject invention may be used in the treatment ofmenstrual cramps, dysmenorrhea, premature labor, endometriosis,tendonitis, bursitis, skin-related conditions such as psoriasis, eczema,burns, sunburn, dermatitis, pancreatitis, hepatitis, lichen planus,scleritis, scleroderma, dermatomyositis, and the like. Other conditionsin which the compounds of the subject invention may be used includediabetes (type I or type II), myocarditis, pathological angiogenesis,and aortic aneurysm.

Moreover, compounds of the subject invention may be used in thetreatment of cardiovascular disease, such as angina, coronary arteryvasospasm, myocardial infarction, coronary ischemia, congestive heartfailure, cardiac allograft vasculopathy, vein graft disease and vascularrestenosis, ischemic reperfusion injury, cerebral artery vasospasm,stroke, cerebral ischemia, essential hypertension, pulmonaryhypertension, renal hypertension and other secondary hypertensivedisorders, atherosclerosis and erectile dysfunction.

The present compounds may also be used in co-therapies, partially orcompletely, in place of other conventional anti-inflammatory therapies,such as together with steroids, NSAIDs, COX-2 selective inhibitors,5-lipoxygenase inhibitors, LTB₄ antagonists and LTA₄ hydrolaseinhibitors. The compounds of the subject invention may also be used toprevent tissue damage when therapeutically combined with antibacterialor antiviral agents.

Differentiated cells produced from hES cells may be useful for treatingdegenerative diseases whose symptoms are caused by loss of a fewparticular cell types. Specific types of neurons have been generatedfrom mouse ES (mES) cells, and similar selective differentiation methodshave been applied to hES cells. However, hES cells have been technicallymuch harder to culture than mES cells, showing problematic propertiessuch as slow growth and insensitivity to the trophic substance leukemiainhibitory factor (LIF). In addition, hES cells are vulnerable toapoptosis upon cellular detachment and dissociation. They undergomassive cell death particularly after complete dissociation, and thecloning efficiency of dissociated hES cells is generally ≦1%. Thus, hEScells are difficult, if not impossible, to use in dissociation culture,which is important for such procedures as clonal isolation followinggene transfer and differentiation induction. Poor survival of humanembryonic stem (hES) cells after cell dissociation is an obstacle toresearch, hindering manipulations such as subcloning.

Recent evidence suggests that addition of selective inhibitors of Rhokinase may enable hES cells to grow and differentiate as mES cells dounder unfavorable culture conditions such as dissociation andsuspension. Rho kinase inhibition has been shown to markedly diminishdissociation-induced apoptosis, increase cloning efficiency (from 1% to−27%) and facilitate subcloning after gene transfer in hES cells. Theimprovement in cloning efficiency conferred Rho kinase inhibition may beparticularly advantageous for isolating relatively rare clones (e.g.,those for homologous recombination) and also for recloning hES cells toobtain a uniform cell quality. Furthermore, dissociated hES cellstreated with selective inhibitors of Rho kinase are protected fromapoptosis even in serum-free suspension (SFEB) culture, form floatingaggregates, and survive and differentiate, as do SFEB-cultured mouse EScells.

Many methods exist for the production or derivation of hES cells. Forexample, histocompatible parthenogenetic human embryonic stem cells(phESC) may be derived from human parthenogenetic blastocysts. Theutility of Rho kinase inhibitors disclosed above, and the methods below,would be expected to be applicable to any hES cells demonstratingtypical hES cell morphology and/or properties, regardless of origin.

Accordingly, the invention contemplates the use of certain compounds andcompositions disclosed herein: for reduction of apoptosis of humanembryonic stem cells; for increasing survival of human embryonic stemcells; for increasing cloning efficiency of human embryonic stem cellsafter gene transfer; and for enhancing differentiation of cultured humanembryonic stem cells. In further embodiments, said prevention ofapoptosis of human embryonic stem cells and/or said increasing ofsurvival of human embryonic stem cells occurs in dissociated culture,such as, for example, serum-free suspension (SFEB) culture.

Besides being useful for human treatment, the compounds and formulationsof the present invention are also useful for veterinary treatment ofcompanion animals, exotic animals and farm animals, including mammals,rodents, and the like. More preferred animals include horses, dogs, andcats.

GENERAL SYNTHETIC METHODS FOR PREPARING COMPOUNDS

The invention is further illustrated by the following examples.

1 NC(═O)c1cc2cccc(CC═C)c2oc1=N 2 Cc1cc(C)c2c(N)c(sc2n1)C(N)═O 3CC(C)c1cc2cc(\C═C\C(O)═O)c(O)cc2o1 4CN(C)CCN(Cc1ccccc1)Cc2cc(ccc2O)-c3ccnc4cc(Cl)ccc34 5CSc1nc(nc(N)c1C#N)-c2ccncc2 6 CC(C)(CO)CNc1nc(cnc1N)-c2ccncc2 7CC(CCc1ccc(O)cc1)Nc2ccc3[nH]ncc3c2 8CC(OC(═O)c1ccc(O)cc1)C(═O)Nc2ccc(Cl)c(Cl)c2 9Nc1nc2ccc(cc2s1)S(═O)(═O)N(CC═C)CC═C 10CC(Oc1ccc2c(C)cc(=O)oc2c1)C(═O)Nc3nccs3 11Oc1ccc(cc1Br)\C═C2\NC(═O)N(Cc3ccccc3F)C2=O 12CCOC(═O)c1nnn(-c2nonc2N)c1-c3ccccc3Cl 13CC1CCC(CC1)C(═O)NC(C(C)C)C(═O)Nc2ccncc2 14Cc1c(C)c2cc(ccc2n1Cc3ccccc3)C(═O)Nc4nc[nH]n4 15Clc1ccc(Cn2cc(cn2)-c3ccncc3)c(Cl)c1

The activity of the compounds in Examples 1-15 as Rho kinase inhibitoris illustrated in the following assay. The other compounds listed above,which have not yet been made or tested, are predicted to have activityin this assay as well.

Biological Activity Assay

In Vitro Rho Kinase Assay

Rho kinase biochemical assays described below depend on fireflyluciferase-based, indirect measurement of total ATP consumption by thekinase following incubation with substrate and ATP. 25 μl of Rho kinaseassay buffer (20 mM Tris-HCL [pH 7.5], 10 mM MgCl₂, 0.4 mM CaCl₂, 0.15mM EGTA, 0.1 mg/ml bovine serum albumin) containing 0.82 μg/ml ofrecombinant N-terminal GST-tagged human Rho kinase 1 (ROCK1, amino acids1-535, Invitrogen Inc., cat. #PV-3691) or recombinant N-terminalGST-tagged human Rho kinase 2 (ROCK2, amino acids 1-552, InvitrogenInc., cat #PV3759), 100 μg/ml S6 peptide substrate (related to aminoacids 218-249 of the human 40S ribosomal protein S6, and suitable forROCK1 or ROCK2, e.g. Upstate/Millipore Inc., cat #12-420), and 3 μM ATPare dispensed to wells of a 384 multi-well opaque plate. The plate iscentrifuged for 30 seconds at approximately 200×g. 240 nl of testcompound in DMSO is dispensed to each well by passive pin transfer. Thelag phase of this in vitro kinase reaction permits addition of compoundssoon after the reaction initiates. The reaction is allowed to incubateat 30° C. for 2 hours. The assay plates are sealed and maintained in ahumidified environment. After 2 hours, 25 μl of easyLite protein kinaseassay reagent (Perkin-Elmer, Inc.) is dispensed. After an additional 10minute incubation at room temperature (about 22° C.), luminescenceactivity is measured on a Molecular Devices Analyst multi-mode platereader or other suitable plate reader. Kinase inhibition results in lessATP consumption, and therefore increased luminescence signal. Negativecontrol activity is measured with DMSO lacking any test compound. Thepositive control is2-methyl-1-(4-methylisoquinolin-5-ylsulfonyl)perhydro-1,4-diazepinehydrochloride (aka H-1152P, HCl salt). Efficacy is measured as apercentage of positive control activity. 50% inhibitory concentration ofcompound (IC50) is measured by assay in dose response. In some cases,kinase reactions and compound testing are performed in 1536 multi-wellplates under similar conditions, with assay volumes appropriatelyscaled.

TABLE 1 Biological Activity ROCK 1 IC₅₀ ROCK 2 IC₅₀ + indicates ≦5 μM +indicates ≦5 μM Example − indicates >5 μM − indicates >5 μM 1 + + 2 + +3 + + 4 − + 5 + + 6 + + 7 + − 8 + + 9 + + 10 − + 11 + + 12 − − 13 + +14 + + 15 + +

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

1. A method of inhibition of Rho kinase, comprising contacting Rhokinase with a compound selected from the group consisting of examples 1to
 15. 2. A method of treatment of a Rho kinase-mediated diseasecomprising the administration of a therapeutically effective amount of acompound selected from the group consisting of examples 1 to 15 to apatient in need thereof.
 3. The method as recited in claim 2, whereinsaid Rho kinase-mediated disease is selected from the group consistingof angina, coronary artery vasospasm, myocardial infarction, coronaryischemia, congestive heart failure, cardiac allograft vasculopathy, veingraft disease and vascular restenosis, ischemic reperfusion injury,transplant reperfusion injury, cerebral artery vasospasm, stroke,cerebral ischemia, essential hypertension, pulmonary hypertension, renalhypertension, a secondary hypertensive disorder, atherosclerosis,bronchial asthma, an acute or chronic obstructive pulmonary disease, anacute or chronic pulmonary inflammatory disease, erectile dysfunction, aneurodegenerative disorder, Alzheimer's disease, multiple sclerosis,brain or spinal cord injury, a disease or trauma-related neuropathy,neuropathic pain, an autoimmune disease, a chronic musculoskeletalinflammatory disease, rheumatoid arthritis, osteoarthritis, a chronicinflammatory bowel disease, Crohn's disease, ulcerative colitis, acuteor chronic inflammatory pain, osteoporosis, a bone disorder, cancer, adisease of pathological angiogenesis, and an ophthalmic disease.
 4. Themethod as recited in claim 3, wherein said Rho kinase-mediated diseaseis an ophthalmic disease.
 5. The method as recited in claim 4, whereinsaid ophthalmic disease is selected from the group consisting ofelevated intraocular pressure and glaucoma.
 6. A method of treatment asrecited in claim 2 further comprising the administration of a secondtherapeutic agent.
 7. A compound selected from the group consisting ofExamples 1 through 15 for use as a medicament.
 8. A compound selectedfrom the group consisting of Examples 1 through 15 for use in themanufacture of a medicament for the prevention or treatment of a diseaseor condition ameliorated by the inhibition Rho kinase.
 9. Apharmaceutical composition comprising a compound selected from the groupconsisting of Examples 1 through 15 together with a pharmaceuticallyacceptable carrier.
 10. The pharmaceutical composition as recited inclaim 9, wherein the pharmaceutical composition is useful for thetreatment or prevention of a Rho kinase-mediated disease.
 11. A methodfor: a. reducing apoptosis of human embryonic stem cells; b. increasingsurvival of human embryonic stem cells; c. increasing cloning efficiencyof human embryonic stem cells after gene transfer; or d. enhancingdifferentiation of cultured human embryonic stem cells, any one of saidmethods comprising the contacting of at least one human embryonic stemcell with an effective amount of a compound selected from the groupconsisting of Examples 1 through 15.